The generation of biologically relevant proteomics data requires samples consisting of homogenous cell populations, in which no unwanted cells of different types and/or development stages obscure the results. The problem is compounded for the analysis of tissue biopsies, since many different cell types are typically present, and small numbers of abnormal cells may lie within or adjacent to unaffected areas. While methods such as laser capture microdissection (LCM) enable the isolation of homogeneous subpopulations of cells, proteomic analysis of LCM-procured specimens is severely constrained by the very low amounts of sample generated. To avoid the limitations of established proteome techniques for analyzing protein extracts obtained from microdissection-procured tissue specimens, an effective discovery-based proteome platform has recently been developed at Calibrant. This proteome platform, called Gemini, combines a unique multidimensional separation system with customized back-end bioinformatics tools, and allows ultrasensitive analysis of minute protein amounts extracted from cells captured by tissue microdissection. This project further aims to employ a novel, laser-free microdissection technique pioneered by our collaborator, Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke (NINDS), capable of providing enriched, high quality, and reproducible tissue samples. By combining Calibrant's ability to perform proteomic profiling from minute samples with the technology and expertise offered by Dr. Zhuang (NINDS) in tissue microdissection and tumor pathology, the proposed research represents a synergistic effort toward the evaluation and validation of a novel biomarker discovery paradigm for enabling the proteome analysis of cancer cells and their micro-environment in support of cancer research, diagnosis, and treatment. Application of the resulting biomarker discovery platform for studying the molecular mechanisms associated with breast carcinoma at the global level will be realized through a collaboration with Professor Fattaneh A. Tavassoli (Yale University School of Medicine), who will apply more than 30 years of research experience in breast cancer pathology and biology and provide access to a collection of fresh frozen human breast cancer biopsies for biomarker discovery. [unreadable] [unreadable] [unreadable]